The derivation of functional cardiomyocytes from human embryonic stem cells (hESCs) fifteen years ago, as well as the discovery of iPSCs, has opened doors to the engineering of human cardiac tissue surrogates for use in drug discovery, disease modeling, and regenerative medicine. Still, translating human iPSC technology to clinical therapy for heart disease has been slow due to a number of challenges including immature and heterogeneous cardiomyocyte phenotype, their low expansion capacity, high metabolic demand and low viability after implantation, potential for tumor and arrhythmia induction, and high costs. To address these limitations, we propose to explore a novel strategy for cell- and gene-based cardiac repair that does not rely on the use of stem cells. Instead, we will develop methods for engineering of terminally differentiated human fibroblasts into cells capable of action potential conduction. These cells will be generated rapidly, at low cost, have stable, homogeneous, and customizable electrical phenotype, be readily expandable in vitro and available off-the-shelf, and be able to electrically couple with cardiomyocytes and significantly improve electrical and contractile function of the infarcted heart. Specifically, in Aim 1 we propose to utilize prokaryotic ion channels to engineer human fibroblasts into a readily expandable and homogeneous source of electrically excitable cells that autonomously fire and conduct action potentials. In Aim 2, we will utilize well-controlled in vitro co-culture systems to explore how engineered fibroblasts with specific electrophysiological properties affect electrical and mechanical function of native cardiomyocytes. In Aim 3, we propose to directly compare actively conducting fibroblasts and PSC-derived cardiomyocytes for their antiarrhythmic action and ability to improve contractile and hemodynamic function of infarcted rat hearts. In addition, we will utilize computer simulations to facilitate genetic engineering of actively conducting fibroblasts and enhance mechanistic understanding of their functional interactions with native cardiomyocytes in vitro and in vivo. We expect that successful completion of this project will enable future applications of engineered fibroblasts in cell-based therapies for myocardial infarction and arrhythmias.